Long-term goal: The long-term goal of this renewal is to unravel the free radical mechanisms by which doxorubicin (DOX), a cancer chemotherapeutic drug that is currently used in the clinic, induces cardiotoxicity in cancer patients.Hypothesis: The general hypothesis to be tested is that DOX-induced apoptosis in cardiomyocytes and endothelial cells is predominantly mediated by hydrogen peroxide (H202) and that antioxidants or antioxidant enzymes that detoxify H202 are antiapoptotic. Specific Aims: First, we will investigate the effect of glutathione peroxidase (GPxI) overexpression on DOX-induced apoptosis in myocytes and endothelial cells. Next, we will demonstrate the role of endothelial nitric oxide synthase (eNOS) in DOX-induced apoptosis. The objective here is to determine whether the DOX/eNOS generated H202 is responsible for apoptosis. DOX-induced generation of reactive oxygen species (ROS) and expression of pro- and antiapoptotic proteins in myocytes and endothelial cells will be assessed using antisense eNOS. Finally, we will determine the mechanism of DOX-induced iron uptake and its implications in apoptosis. After establishing the role of ROS and eNOS in DOX-induced apoptosis, we will investigate the mechanism of antiapoptotic action of selected metalloporphyrins in this system. Methods: We will use cardiomyocytes isolated from adult rat hearts, cultured bovine aortic endothelial cells, and adenovirally transfected GPx1 overexpressing cells. Apoptosis will be detected by several methods including TUNEL analysis, caspase activity, mitochondrial cytochrome c release, Bcl-2 and Bax activity. Superoxide levels will be determined by fluorescence and spin-trapping. We will use the state-of-the-art ESR technique to detect the various redox states of metalloporphyrins. Significance: Drug toxicity is a serious side effect of cancer chemotherapy and severely limits the clinical usefulness of most widely used drugs such as doxorubicin. Children treated with DOX for leukemia develop cardiomyopathy years after cessation of DOX chemotherapy. Further understanding of the oxidative mechanisms may lead to an effective antioxidant/antiapoptotic therapy for minimizing cardiotoxicity. Novelty: Emerging literature indicate that cardiomyocyte apoptosis contributes to heart failure. Understanding the relationship between DOX-induced apoptosis and ROS formation may help discover novel antioxidant/antiapoptotic therapy in oxidant-induced disease processes.